Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Autores
Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines
Año de publicación
2023
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.
Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Fil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Fil: Manifava, Maria. The Babraham Institute; Reino Unido
Fil: Ktistakis, Nicholas. The Babraham Institute; Reino Unido
Fil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Materia
AUTOPHAGY
LC3
INMUNOFLUORESCENCE
PANCREAS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/227937
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/227937
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 ImmunofluorescenceRenna, Felipe JavierHerrera Lopez, MalenaManifava, MariaKtistakis, NicholasVaccaro, Maria InesAUTOPHAGYLC3INMUNOFLUORESCENCEPANCREAShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Manifava, Maria. The Babraham Institute; Reino UnidoFil: Ktistakis, Nicholas. The Babraham Institute; Reino UnidoFil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaJournal of Visualized Experiments2023-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/227937Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines; Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence; Journal of Visualized Experiments; Journal of Visualized Experiments; 194; 4-2023; 1-131940-087XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.jove.com/video/65005info:eu-repo/semantics/altIdentifier/doi/10.3791/65005info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:07:34Zoai:ri.conicet.gov.ar:11336/227937instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:07:34.86CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
title Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
spellingShingle Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
Renna, Felipe Javier
AUTOPHAGY
LC3
INMUNOFLUORESCENCE
PANCREAS
title_short Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
title_full Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
title_fullStr Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
title_full_unstemmed Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
title_sort Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
dc.creator.none.fl_str_mv Renna, Felipe Javier
Herrera Lopez, Malena
Manifava, Maria
Ktistakis, Nicholas
Vaccaro, Maria Ines
author Renna, Felipe Javier
author_facet Renna, Felipe Javier
Herrera Lopez, Malena
Manifava, Maria
Ktistakis, Nicholas
Vaccaro, Maria Ines
author_role author
author2 Herrera Lopez, Malena
Manifava, Maria
Ktistakis, Nicholas
Vaccaro, Maria Ines
author2_role author
author
author
author
dc.subject.none.fl_str_mv AUTOPHAGY
LC3
INMUNOFLUORESCENCE
PANCREAS
topic AUTOPHAGY
LC3
INMUNOFLUORESCENCE
PANCREAS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.
Fil: Renna, Felipe Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Fil: Herrera Lopez, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
Fil: Manifava, Maria. The Babraham Institute; Reino Unido
Fil: Ktistakis, Nicholas. The Babraham Institute; Reino Unido
Fil: Vaccaro, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina
description Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.
publishDate 2023
dc.date.none.fl_str_mv 2023-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/227937
Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines; Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence; Journal of Visualized Experiments; Journal of Visualized Experiments; 194; 4-2023; 1-13
1940-087X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/227937
identifier_str_mv Renna, Felipe Javier; Herrera Lopez, Malena; Manifava, Maria; Ktistakis, Nicholas; Vaccaro, Maria Ines; Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence; Journal of Visualized Experiments; Journal of Visualized Experiments; 194; 4-2023; 1-13
1940-087X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.jove.com/video/65005
info:eu-repo/semantics/altIdentifier/doi/10.3791/65005
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Journal of Visualized Experiments
publisher.none.fl_str_mv Journal of Visualized Experiments
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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